Approximately one third of the world""s population is infected with Mycobacterium tuberculosis (MTB), the causative agent of human tuberculosis (TB). MTB is responsible for 2-3 million deaths annually, giving it the dubious distinction of being the leading cause of death due to a single infectious agent. In addition, TB ranks seventh in causes of global mortality and disability, and if current predictions prove correct, it will remain among the top 10 causes of disease, well into the next century (Murray and Lopez, Lancet, 349:1498-1504 (1997)). Directly observed treatment, short-course (DOTS) is the tactic proposed by the World Health Organization (WHO) to control the global TB crisis (Murray and Solomon, PNAS USA, 95:13881-13886 (1998)). DOTS has proven to be an effective strategy in several national TB control programs, with cure rates approaching 90% (Lancet, 347:358-362 (1996)). However, since global implementation of DOTS programs is occurring at a slow-moving pace, it is likely that additional measures will be needed to stem the tide of TB mortality. It has been estimated that the introduction of a new vaccine of only 50% efficacy could decrease the incidence of TB by 36 million cases, saving 9 million lives (Murray and Salomon, 1998). Thus, by coupling efficacious vaccination with effective treatment, greater success in global TB management would be anticipated.
Bacille Calmette-Guerin (BCG), an attenuated strain of M. bovis, is the currently available vaccine for the prevention of tuberculosis. It was created empirically by repeated passage in the laboratory, and for reasons that are as yet undefined, it is avirulent in immunocompetent hosts. In several animal models of infection, BCG has been demonstrated to induce protective immunity against MTB. Since its implementation in 1928 as a TB vaccine, more doses of BCG have been administered than any other vaccine, as an estimated 3 billion people have received BCG vaccination for the prevention of tuberculosis. Although the use of BCG is unquestionably safe in immunocompetent individuals, it has shown itself to be of variable efficacy. While in certain populations, vaccination with BCG has been highly effective in preventing tuberculosis, in others it has failed miserably. In the largest clinical trial that took place in India involving more that 100,000 persons, BCG exhibited a calculated protective efficacy of zero. Thus, the generation of an improved vaccine(s) to replace BCG and to prevent tuberculosis is urgently needed.
Relative to wildtype M. tuberculosis, 15-16 regions of the MTB genome are not represented in BCG. Eleven of these segments cannot be found even in virulent strains of M. bovis; of the remaining 5, 4 are missing from all BCG strains examined. It is probable that one or more of the 38 open reading frames (ORFs) specifically missing from BCG are required for virulence. Of interest, is the finding that a number of predicted transcriptional regulators identified by the H37Rv genome sequencing project (Cole, et al., Nature, 393:537-544 (1998)) would be located in these BCG deletions. The loss of a regulatory protein would be expected to affect multiple genetic loci and could lead to deranged gene expression in vivo. Consistent with this hypothesis, is the demonstration that reintroduction of one of these deleted regions into BCG results in the repression of at least 10 proteins and the upregulated expression of others. It is conceivable that potentially immunogenic and immunoprotective antigens might be missing from or inappropriately expressed in BCG, and therefore, compromising the immune response generated from this vaccine. For example, it has been noted that the gene for ESAT 6, a highly immunogenic, secreted protein of M. tuberculosis, is located within one of these deleted chromosomal regions. It has been demonstrated that protective immunity against experimental tuberculosis can be provided by prior immunization with supernatants containing a mixture of MTB secreted antigens, of which ESAT 6 is one. It is possible, that if one or more of the proteins encoded within the deleted regions were present at vaccination, the immune response elicited might be more efficacious.
It also has been demonstrated in both mice and guinea pigs that primary infection with MTB will induce resistance to re-infection (Dubos and Schaefer, Ann. Rev. Tuberculous Pulmonary Dis., 74:541-551 (1956); Kanai and Yanagisawa, Jap. J. Med. Sci. and Biol., 8:115-127 (1955)). Thus, it is feasible that a vaccine could be created by the rational deletion of genes of MTB, promoting the attenuation of, but preserving the immunogenicity and protectiveness of the bacillus. Ideally, such a vaccine would provide better protection than BCG.
An approach to the creation of vaccine strains that are avirulent yet immunogenic that has proved to be successful in other systems is the introduction of mutations conferring auxotrophy, or specific requirements for growth. For example, aromatic amino acid auxotrophs of Salmonella typhimurium, Shigella flexneri, Bordetella pertussis, Yersinia spp., Pasteurella multocida, Bacillus anthrasis and Corynebacterium pseudotuberculosis (Simmons, et al., Infect. Immun., 65:3048-3056 (1997)) have displayed reduced virulence while exhibiting protection in various in vivo experimental systems. Similarly, purine and pyrimidine auxotrophs of Salmonella spp are attenuated in vivo. In addition, Bacon and colleagues created a strain of S. typhimurium that required exogenous leucine for growth and showed that the strain had diminished virulence for mice (Bacon, et al., Brit. J. Exp. Path., 31:714-723 (1950)). Likewise, a leucine auxotroph of BCG, created by insertional disruption of leuD, a gene essential for leucine biosynthesis, failed to grow in either immunocompetent (McAdam, et al., J. Clin. Invest., 66:441-450 (1995)) or immunodeficient SCID mice (Guliera, et al., Nature Med., 2:334-337 (1996)).
The present invention provides a recombinant M. tuberculosis mycobacterium that is auxotrophic for leucine. The present invention also provides a vaccine comprising a recombinant M. tuberculosis mycobacterium that is auxotrophic for leucine, as well as a method for treating or preventing tuberculosis in a subject comprising administering to the subject a recombinant M. tuberculosis mycobacterium that is auxotrophic for leucine in an amount effective to treat or prevent tuberculosis in the subject. Additional objects of the present invention will be apparent from the description which follows.